Formation of Zinc Protoporphyrin IX and Protoporphyrin IX from Oxymyoglobin in Porcine Heart Mitochondria

Abstract

The formation of zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PP) in porcine heart mitochondria was investigated by fluorescent spectral analysis. The time-course study at -1῍C and pH /./ with +** mM ZnCl, showed a linear formation of ZnPP from oxymyoglobin after a lag-period (3 h) at the formation rate of *..0 nmol/h. The formed amount of ZnPP is dependent upon the ZnCl, concentration. In the incubation without ZnCl,, the new peak derived from PP appeared at 0--nm after +, h, and then its intensity gradually increased. The formation of PP is believed to be due to the release of Fe ,ῌ in porphyrin ring of oxymyoglobin during the incubation. This suggests that the mitochondria have the ability to form PP from oxymyoglobin, and therefore, are directly related to the release of Fe ,ῌ from porphyrin ring in myoglobin.

Color Formation in Nitrite‐Free Dried Hams as Related to Zn‐Protoporphyrin IX and Zn‐Chelatase Activity

“…Investigations by several researchers into the role of Zn‐chelatase in ZPP formation were useful to elucidate its pivotal 2‐step function in removing the iron from heme then encouraging zinc insertion into the resulting free protoporphyrin moiety (Ishikawa and others 2007; Taketani and others 2007). However, there was little or no focus on the enzyme activity as affected by chemical changes occurring in the cured meat nor was it clarified whether the enzyme remains active throughout the lifespan of dried hams, which is of 1 y and more.…”

“…Working with rat liver as a substrate, Taketani and others (2007) isolated and identified the enzyme which promotes ZPP synthesis as mitochondrial Fe‐chelatase and they argued that both iron removal and zinc insertion would be favored by this unique enzyme, with the former step being the time limiting one or the lag‐phase of the reaction, as described also by Ishikawa and others (2007) in a time‐course study of ZPP synthesis from porcine heart extracts. Therefore there is evidence that zinc‐protoporphyrin IX would form in dried meat as a result of an all‐natural reaction where substrates are delivered by the muscle itself, including the enzyme taking part in the 2‐step synthesis of ZPP.…”

Porcine Ferrochelatase: The Relationship between Iron-Removal Reaction and the Conversion of Heme to Zn-Protoporphyrin

“…13) We and others have reported that the reverse reaction of ferrochelatase, namely, removal of iron from heme, occurs in vitro. 10,16) Therefore, hemoprotein-heme, including myoglobin and hemoglobin, becomes a substrate of the removal reaction of FECH, 10) and the protoporphyrin thus produced can be utilized in the formation of Zn-protoporphyrin, a major pigment of dry-cured ham. The enzyme naturally utilizes ferrous ions as a substrate in vivo, but additionally inserts divalent metal ions such as zinc and cobaltic ions into porphyrin rings in vitro.…”

“…Immunoblotting was carried out using anti-ferrochelatase as primary antibody. 16) Enzyme assay. FECH activity was determined by measuring the insertion of zinc into mesoporphyrin, as described previously.…”

“…15) Although the mechanism involved in the formation of Zn-protoporphyrin during the production of dry ham is unclear, formation in the muscle may be related to mitochondria and enzyme catalysis. 16) Very recently, we found that FECH is involved mainly in the formation of Zn-protoporphyrin via iron-removal reverse reaction, 10) but little attention has been paid to the characteristics and kinetic properties of the reverse reaction of FECH.…”